Published studies from several labs, including our own, have demonstrated essential roles for the orphan nuclear receptor, Steriodigenic factor 1 (SF-1) in endocrine organogenesis, male sexual differentiation, and steroid homeostasis. Our prior work shows that maximal SF-1 mediated transcription and recruitment of general cofactors depends on a single phospho-serine 203 located in the hinge region, and that receptor phosphorylation mimics several features of ligand-dependent receptor activation. Here, we propose to: Aim 1 Determine the in vivo role of SF-1 serine 203 phosphorylation. Aim 2 Identify cofactors that are recruited upon SF-1 phosphorylation by using a yeast genetic screen, an affinity-GST purification method, or a high-throughput peptide binding assay. Aim 3 Establish the function and dynamics of cofactor recruitment for identified candidates. The potential role for phosphorylation in regulating nuclear receptor activity has been provocative, but remained secondary to ligand-activation of receptors. Now, because some members of the nuclear receptor gene family may not have specific high affinity ligand, post-translational events and protein-protein interactions have assumed a more legitimate position in modulating receptor activity. To date, studies addressing how extracellular signaling and nuclear receptor function are integrated have been limited to cellular model systems. As such, our aim to assess the in vivo function of SF-1 phosphorylation will be of significance. Our preliminary data show that SF-1 is phosphorylated at S203 in endocrine tissue; these observations provide a strong rationale for using mouse genetics to determine the in vivo significance of this modification. We are also excited by our new finding that the UBC-9 SUMOylation conjugating enzyme interacts with SF-1. Indeed, the close proximity of the MAPK phosphorylation site to the perfect SUMOylation consensus site in the hinge region of SF-1 is provocative, and warrants further investigation into the possible interplay between these two posttranslational events. We believe our studies with SF-1 will provide valuable insights into how posttranslational events modulate ligand-independent receptor activation and will be directly relevant to the progression of hormone-insensitive endocrine tissue cancers.